ABSTRACT Down syndrome (DS) or trisomy 21 is associated with multiple developmental anomalies affecting the cardiovascular, hematological, respiratory, immunological, endocrine, gastrointestinal and neurological systems. The mechanisms by which an additional copy of chromosome (Chr) 21 produces these systemic issues remains poorly understood. We have previously recruited 600 DS patients with congenital heart defects (CHD) and 100 DS patients without CHD. Using analyses of discarded CHD tissues obtained during surgical repair, we identified unexpected and distinct cardiac gene expression differences between euploid and DS CHD patients. Included among these we found that SOST (encoding sclerostin on Chr 17) expression was 10-fold higher in DS than in euploid CHD tissues. As sclerostin inhibits WNT and BMP signaling, we hypothesis that dysregulation may contribute to CHD in some DS patients. Sclerostin also impact bone density and may contribute to skeletal and other abnormalities in DS. We propose to expand these analyses to define genetic basis for abnormal gene expression that influence DS phenotype. We hypothesize that the molecular response to trisomy 21 are influenced by additional genotypes that result in patient-specific DS phenotypes. We will define these genotypes and their related regulatory networks that alter cell and organ development. We will capitalize on whole genome sequences (WGS) from ~700 DS patients with different DS phenotypes that will be available for the proposed studies. We are generating induced pluripotent stem cells (iPSCs) from 200 DS patients. WGS and iPSC derivation are supported by other funding mechanisms. We propose to analyze WGS to define sequence elements associated with DS phenotypes. We will explore the transcriptional and epigenetic consequences of these associations using DS iPSCs and cell lineages differentiated from iPSCs. Through the differentiation of DS iPS cells into cardiomyocytes (iPSC-CMs), endothelial, neural cells and others will define the consequences of sequences on gene expression. We will also use enhanced CRISPR/ Cas9 engineering and single cell analyses of iPSCs to investigate the gene regulatory mechanisms associated with DS phenotypes. We will also explore if the consequences of DS- associated variants with in euploid cells, to determine if trisomy is required conditioning genotype. We propose to validate finding using DS patient iPSCs and discarded euploid and trisomy 21 cardiac tissues. To achieve these broad goals, we will: 1) Identify genetic variants associated with DS phenotypes using whole genome sequence and RNA expression eQTLs. 2) Compare single cell/nucleus transcriptional and ATACseq profiles of DS and euploid iPSCs, differentiated cells, and CHD tissues. 3) Define genes and regulatory pathways that modulate DS phenotypes by perturbing gene expression in DS iPSCs and cell lineages.